Objective To investigate the cytotoxicity of a conditionally replicative adenovirus (CRAd) combined with the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene system delivered to Y79 cells. Methods Experimental study. Selective replication of CRAd armed with HSVtk, i.e., Ad-hTERT-E1A/CMV-HSVtk, in Y79 cells was observed after Y79 and human retinal pigment epithelial (hRPE) cells were infected with CRAd at 0 h, 24 h, 48 h and 72 h.Y79 cells were divided into 5 groups, an untreated group, a GCV treatment group, a CRAd treatment group, a dl309 group and a CRAd plus GCV treatment group, for studying the cytotoxicity of CRAd combined with HSVtk/GCV system. At 48 h post infection with the viruses, the medium of the cells was replenished with a culture medium with or without 10 μg/ml of GCV. At 3 days post infection with the viruses, the cytotoxicity of CRAd combined with the HSVtk/GCV suicide gene system delivered to Y79 cells was observed with bioluminescent imaging (BLI) and the Cell Counting Kit-8 (CCK-8). Results CRAd could selectively replicate in Y79 cells. At 72 h post infection, viral titer increased by more than 100 fold in Y79 cells infected with CRAd. CRAd showed no cytotoxicity to Y79 cells at doses of no more than 10 MOI. It showed cytotoxicity to Y79 cells with about 20%and 30% of cell death with viral doses at 50 and 100 MOI, respectively. When combined with GCV, the cytotoxic effect of CRAd was amplified significantly. CRAd plus GCV could lead to about 50% and 80% of Y79 cell death with viral doses at 50 and 100 MOI, respectively. Conclusion CRAd has the ability of tumor-specific replication in Y79 cells. The combination of CRAd with HSVtk/GCV can effectively lyse Y79 cells in vitro.